In vitro antibacterial effects of silver nanoparticles synthesized using Verbena officinalis leaf extract on Yersinia ruckeri, Vibrio cholera and Listeria monocytogenes.

Background and Objectives
The use of plants for the synthesis of nanoparticles has received attention. The present study aimed to evaluate the antibacterial effects of silver nanoparticles synthesized by Verbena officinalis leaf extract against Yersinia ruckeri, Vibrio cholerae and Listeria monocytogenes.


Materials and Methods
Silver nanoparticles were obtained by reacting silver nitrate solution 2 mM and V. officinalis leaf extract. The AgNPs were characterized by UV-visible spectrophotometer, scanning electron microscopy (SEM), and Fourier transform infrared spectrometer (FTIR). To determine minimum inhibitory concentration and test antibiogram of nanoparticle synthesized, broth micro dilution and agar well diffusion methods were used, respectively.


Results
The zones of bacterial inhibition were 16 ± 0.5 and 9.16 ± 0.28 mm against Y. ruckeri and L. monocytogenes using 10 and 0.62 mg/mL AgNPs, respectively. Among the studied bacterial species, silver nanoparticles were more effective on Y. ruckeri and L. monocytogenes and less effective on V. cholerae. The highest MIC and MBC of AgNPs (2.5 and 5 mg/mL) were observed for V. cholerae. The lowest MIC and MBC of AgNPs (0.32 and 0.62 mg/mL) were observed for Y. ruckeri, respectively. The MIC and MBC of AgNPs were found to be 1.25 and 2.5 mg/mL for L. monocytogenes.


Conclusion
The results clearly indicated that V. officinalis AgNPs have potential antimicrobial activity against Gram-positive and Gram-negative bacteria.


INTRODUCTION
Nanotechnology is a general term that refers to all advanced technologies in the field of working with nanoscale. Usually the purpose of nanoscale dimension is about 1 nm to 100 nm (1). Nano-materials based on metal ions have wide cytotoxicity activity against bacteria, fungi and viruses. Nano materials and specially, nano-metal materials, due to having a superficial load and the ratio of surface to volume, disable enzymes and DNA of microorganisms with electron balance between groups of electron donor, such as, thiol, carboxylate, amide, imidazole, indole and hydroxyl (2). Various methods are used for producing nanoparticles, such as reactions of chemicals and photochemicals in reverse micelles, thermal analysis compounds with the help of radiation, electrochemical methods, sonochemical, processing with microwaves. Unfortunately, the use of hazardous substances is inevitable to produce nanoparticles in most methods (3). The disadvantages of synthesis methods of nanoparticle are the use of harmful toxic chemicals, low conversion rates, high consumption of materials and energy. Thus, for overcome these limitations, the growing demand for the development of safe and environment-friendly process of the synthesis of nanoparticles is required.
Green synthesis techniques or biological methods that use microorganisms, such as fungi, bacteria, or plant extracts, are an alternative method of simple causative agent of enteric red mouth disease, especially rainbow trout and atlantic salmon fish (14). Infections due to Y. ruckeri cause high mortality in fish aquaculture systems, leading to significant economic losses in the fish farming industry (15). The bacterium is shed in the faeces of infected fish and the disease can be transmitted by water. Listeria monocytogenes is a Gram-positive foodborne pathogen that is ubiquitously found in diverse environments, such as soil, water, various food products, animals and humans (16), which is often transmitted through contaminated water and food (17). Vibrio cholerae is a facultative human pathogen found in coastal waters and causes acute gastrointestinal disease, cholera, a major health threat in poor nations (18). V. cholerae causes severe diarrhea in humans (19).
In this study, V. officinalis leaf extract was used to convert the silver ions to silver nanoparticles by silver nitrate solution. The antibacterial effects of silver nanoparticles synthesized by V. officinalis leaf extract was evaluted against Y. ruckeri, V. cholerae and L. monocytogenes.

Preparation of material and microorganisms.
Leaf of V. officinalis were obtianed from the farms growing medicinal plants at the agricultural research institute located at Baghiatoallah Azam Complex of and practical for physical and chemical methods.
University of Zabol in Zabol. AgNO and Nutrient The use of plant extracts for biological synthesis of nanoparticles is an important method. It is rapid, cost-effective, environment-friendly and has received attention (4). So far, the biosynthesis of silver nanoparticles by plants, such as Artemisia nilagirica (5), Piper longum (6), Acalypha indica (7), and many others, has been conducted. Verbena officinalis is perennial herb native to Europe. It is used in folk medicine for the treatment of inflammatory disorders (8,9), gastric diseases, abrasions, and skin burns (10,11). It is commonly used for biological activity, pharmacokinetic effect, antitumor, antioxidant and antifungal activity, antiradical efficacy, analgesic activity, neuroprotective effects, anti-inflammatory activity, and antioxidant activities (12). A recent survey indicated that the main ingredients in the essential oil of V. officinalis are citral, geranial, neral, geraniol, limonene and cineole (13). The 3 bacteria studied were selected because Yersinia ruckeri is the agar (NA) were purchased from Merck Company. The microorganisms used in this experiment were Listeria monocytogenes (1298), Yersinia ruckeri (KC291153), and Vibrio cholerae (1611).
Preparing plant extracts and synthesis of silve nanoparticle. To prepare aqueous extracts of V. officinalis, first, leaves of V. officinalis were washed 2 times, once with purified water and once with distilled water. The leaves were air-dried under shade and powdered using a disintegrator. About 2 grams of the sample was added to 20 mL sterilized distilled water and was placed in a shaker incubator for 2 hours. The extract was cooled and filtered using Whatman no1 filter paper (20). For the synthesis of silver nanoparticle, the stocked silver nitrate solution 0.1 M (0.169 g in 10 mL of distilled water) was prepared. Synthesis of silver nanoparticles was done by mixing and reaction of silver nitrate solution of 2 mM and extract of V. officinalis (1 mL of the extract was obtained by 19 mL distilled water and combined; then, 400 μL from silver nitrate solution of 0.1 M was added). Next, the mixture was placed in the incubator at a temperature of 37°C for 24 hours.
Characterization of silver nanoparticles, UV-Vis spectra analysis. Sample (1 mL) of the suspension was collected periodically to monitor the completion of bioreduction of Ag+ in aqueous solution, 1 mL of sample was diluted with 2 mL of distilled water; then, the UV-Vis spectrum of solution was measured between wavelength 280 to 700 nm in a spectrophotometer (Rayleigh, UV-2100, China), with a resolution of 1 nm.
Fourier transform infrared spectroscopy analysis. The dried Ag NPs were analyzed using the potassium bromide (KBr) pellet (FTIR grade) method in 1:100 ratio and the spectrum was recorded using FTIR (Bruker optics Ft Tensor, 27, Germany).
Scanning electron microscopy analysis. AgNPs was centrifuged at 10000 rpm for 15 minutes. Supernatant was collected and poured nanoparticles were deposited on glass slides and dried at room temperature. The images of AgNPs were obtained using a scanning electron microscope (KYKY, Model No. EM-3200).
Evaluation of antibacterial effects of silver nanoparticles: Minimum inhibitory concentration. Y. ruckeri, V. cholerae and L. monocytogenes bacteria were grown in nutrient broth (NB) medium. Concentrations of 10, 5, 2.5, 1.25, 0.62, 0.31 mg/mL synthesized silver nanoparticles were used to evaluate the antibacterial effects on Y. ruckeri, V. cholerae and L. monocytogenes. Concentrations were determined by broth micro-dilution technique in sterile 96 well plate. A volume of 100 μL of nanoparticles synthesized at concentration of 20 mg/mL was placed into the first well of the plate microtiter that contained 100 μL NB medium to obtain a concentration of 10 mg/mL. Serial dilution was performed by pumping the contents of the first well and removal of 100 μL from it and adding it to the second well. This operation was done to the last well. Then, 100 μL of bacterial suspension, equivalent to 0.5 mcFarland (1.5 × 10 6 CFU/ mL), was added to the wells. Each plate was prepared with a set of controls. Plates were placed in an incubator (Binder, USA) at 37°C for V. cholerae and L. monocytogenes and 30°C for Y. ruckeri for 18-24 hours. The lowest concentration at which no visible bacterial growth could be found was taken as the MIC value (21).
Minimum bactericidal concentration. The method used and described below is an amended version of the procedure described in the BSAC Guide to Sensitivity Testing and can be adapted for determining the minimum bactericidal concentration (MBC) of silver nanoparticles synthesized by biological method. After determining the MIC of silver nanoparticles synthesized, 10 μL from all wells that had no visible bacterial growth was removed and was then cultured on the NA media. Next, the plates were incubated for 1 night at 37°C for V. cholerae and L. monocytogenes and 30°C for Y. ruckeri. MBC is the lowest concentration of antimicrobial agent that openly kills >99.9% of the initial bacterial population where no visible growth of the bacteria is observed on NA medium (21).
Antibacterial activity by agar well diffusion method. Antibacterial activity of the synthesized AgNPs was analyzed using the agar well diffusion assay method. The 20 mL medium of mueller-hinton agar (MHA) semi-solid was poured into the petri dishes. The bacteria were grown in NB for 24 hours and by sterile swab on the surface of solid medium MHA and were cultured with 1.5 × 10 6 CFU/mL suspensions of test bacteria. Different concentration of silver nanoparticles (ranging from 0.32 to 10 mg/ mL) was impregnated into well (6 mm diameter) and placed on the surface of inoculated agar plates. Plates were incubated for 24 hours at 37°C for V. cholerae and L. monocytogenes and 30°C for Y. ruckeri. Antimicrobial activity was measured based on the diameter of inhibition zone in mm. Statistical analysis was performed using one-way analysis of variance with Tukey's test, which is used to compare the differences among samples. P values ≤ 0.05 were considered significant, and all antibacterial assays were performed with 3 replications.

RESULTS
AgNPs characterization: UV-vis spectra analysis. As the V. officinalis leaf aqueous extract was http://ijm.tums.ac.ir SILVER NANOPARTICLES SYNTHESIZED USING VERBENA OFFICINALIS 3 added to silver nitrate solution, the color of the solution changed from light yellow to reddish brown after the process of reduction of Ag+ to Ag nanoparticle, which indicated AgNPs formation (Fig. 1). Fig.  2 shows the UV-visible spectra of silver nanoparti-comparison with AgNPs, no antimicrobial activity was observed in the aqueous extract of plant (control).
Minimum inhibitory concentration and minicle formation using constant AgNO concentration mum bactericidal concentration. The results of the (2 mM) with V. officinalis leaf extract at 37°C after 24 hours between wavelength of 280 to 700 nm. The results of optical density showed that the maximum absorption measuring. solution containing the nanoparticles was at around 420 nm. Scanning electron microscopy analysis. The SEM image of the AgNPs is displayed in Fig. 4. Nanoparticles were formed with an average size of 42.57 ± 5.34 nm.
Antibacterial property analysis. In this study, the antimicrobial property of AgNPs was investigated by growing Y. ruckeri, V. cholerae and L. monocytogenes colonies on MHA plates, supplemented with different concentrations of AgNPs. Results obtained are shown in Table 1 and Fig. 7. The inhibition zones obtained indicate maximum antibacterial activity of the prepared test sample. The zone of bacterial inhibition by AgNPs prepared from V. officinalis leaf extract shows maximum inhibition for Y. ruckeri at 10 mg/mL, with 16 ± 0.5 mm and minimum inhibition for L. monocytogenes at 0.62 mg/mL, with 9.16 ± 0.28 mm. Also, in MIC and MBC of nanoparticles synthesized on the Y. ruckeri, V. cholerae and L. monocytogenes bacteria are shown in Table 2

Growth of bacteria [+] and the inhibition of bacterial growth [−]
The present study investigated the synthesis of nanoparticles and the methods used for the diagnosis http://ijm.tums.ac.ir NARJES SANCHOOLI ET AL. 3 3 and evaluation of antibacterial effects of nanoparticles by V. officinalis extract. In this study, after 24 hours of the conversion process, silver nanoparticle showed reddish-brown color, suggesting the formation of silver nanoparticles in solution and, in fact, inhibition for Y. ruckeri at 10 mg/mL AgNPs, with 16 ± 0.5 mm, followed by L. monocytogenes (15.16 ± 0.57) and V. cholerae (13.16 ± 0.28). In a study, Kumarasamyraja and Jeganathan (2013) (20) showed that the maximum zone of inhibition at concentration confirmed the reaction between AgNO and V. of-of 300 μg/mL of Acalypha indica Ag-NPs, with avficinalis leaf extract, which is similar to previous studies (4,24,25). In the present study, the size of silver nanoparticles synthesized using V. officinalis extract was obtained to be 33-49 nm. The synthesis of nanoparticles, with a size of 10-45 nm, using extracts of Vitis vinifera, was reported by Roy et al. in 2013 (26). In another study, silver nanoparticles, with an average size of 13-57 nm, were synthesized using extract Ixora coccinea (27). Green synthesized silver nanoparticles by V. officinalis leaf extract, with average size of 42.57 nm, were examined for their antibacterial activity using selected pathogens. In the present work, MHA well diffusion test was performed for antibacterial activity on Gram-positive bacteria, such as L. monocytogenes and Gram-negative bacteria, such as Y. ruckeri and V. cholerae. In the study of Mariselvam et al. (2014) (28), the zones were used to determine the activeness of the synthesized nanoparticles. The zone with the diameter of <9 mm was considered as inactive; 9-12 mm as partially active, and 13-18 mm as active. In this study, most of the AgNPs samples were active against Y. ruckeri and L. monocytogenes and less active against V. cholerae. In the present study, the effects of nanoparticles synthesized on Gram-positive and Gram-negative bacteria were almost identical. Jain et al. (2009) (29) studied the antibacterial effects of silver nanoparticles synthesized by papaya plant on E. coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria and their results were similar to those of the present study. The antimicrobial effects of silver nanoparticles on both Gram-positive and Gram-negative bacteria were evaluated and attributed to the same effects of silver nanoparticles on the wall and membrane of bacteria (30). Many sources have pointed to the deadly effects of silver nanoparticles due to simultaneous activity on the wall, the ability to penetrate the cell membrane, and the effect on cell respiration chain, RNA and DNA; these structures are the same in Gram-positive and Gram-negative. Thus, the antibacterial properties of silver nanoparticles in both groups of bacteria were approximately the same (31).
Our experiments revealed that there is maximum erage particle size of 0.516 μm on the P. aeruginosa, were 16 mm, followed by E. coli (14 mm (33). Bactericidal activity by silver nanoparticles depends on the concentration of AgNO . For example, if the metal concentration is lower, its antibacterial activity is more, and if the metal concentration is higher, its activity is less. The smaller particles have greater level of interaction, so they have more antibacterial effect compared to larger particles (34). The mechanism for the inhibition zone diameter may be due to the increase in proton motive force taking place on the surface of the bacteria due to ionic bond formation with AgNPs (35). Cell membranes of microorganisms are negatively charged and silver nanoparticles are positively charged; and when the charges of these particles come together, significant conformational changes are made in the membrane and the membrane eventually loses control of its permeability, leading to death of the cell (36). The MIC of the AgNPs sample, which effectively inhibits the growth of tested pathogens, is called MIC value of that particular sample.  (37) also studied the biosynthesis of silver nanoparticles by onion extract against E. coli and Salmonella Typhimurium; in their study, concentrations of 50 μg/mL confirmed an effective concentration. MIC in the above-mentioned studies was less than the value obtained in this study. Studies by Jain and Sharma (2013) also showed that it is possible to inhibit the growth of yeast at low concentrations of silver nanoparticles (30). Silver nanoparticles are most effective in preventing mitochondrial activity, and cell death occurs with the production of lytic enzymes. Nanoparticles show new or improved properties based on specific characteristics, such as size, distribution, and morphology (38). In addition to particle size, particle shape plays an important role in the antimicrobial properties of nanoparticle; formless angular nanoparticles have the most properties and spherical and rod-shaped particles have the least properties, respectively (39). The low properties of this study may be due to the shape of the rod and the large particle size. The results clearly indicated that V. officinalis AgNPs have potential antimicrobial activity against Gram-positive and Gram-negative bacteria.